{"paper_id":"2fc5a7e2-e830-4c98-aa34-283956345b7c","body_text":"Harpagide Alleviates Cerebral Ischemic Injury via AMPK-MCU Phosphorylation-Dependent Inhibition of Mitochondrial Calcium Overload | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Harpagide Alleviates Cerebral Ischemic Injury via AMPK-MCU Phosphorylation-Dependent Inhibition of Mitochondrial Calcium Overload Ke Wang, Huai-yu Liu, Yue Wang, Geng-zheng Su, Jin-lai Gao, Yan-ping Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8705254/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Mitochondrial calcium homeostasis may offer therapeutic benefits for ischemic stroke. Harpagide has been shown to inhibit mitochondrial calcium uptake, but the mechanism remains unclear. In this study, a male ICR mouse model of transient middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia and PC12 and SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) were used. Apoptosis, lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), and the ATP levels were measured to assess the neuroprotective effects of harpagide. DCFH-DA, JC-1, and Rhod-2A probes were used to analyze the mitochondrial function. RT-qPCR and western blot were used to determine the mitochondrial calcium uniporter (MCU), the apoptosis-inducing factor (AIF), Endo G, Cyt C, Caspase-3, and AMPK-MCU phosphorylation expression levels. Additionally, Longa scoring and triphenyl tetrazolium chloride (TTC) staining were used. Transmission electron microscopy (TEM) was used to examine the mitochondrial ultrastructural changes. An immunohistochemical analysis was conducted to detect the MCU expression in the temporal cortex. We found that harpagide exhibits robust neuroprotective effects both in vivo and in vitro , decreased LDH release and MDA levels while increasing SOD activity and ATP content. Harpagide also inhibited mitochondrial calcium overload, mitigated mitochondrial damage, and regulated the MCU, AIF, Endo G, Cyt C, and Caspase-3 expression to reduce mitochondria-mediated apoptosis. Mechanistically, harpagide exerted its neuroprotective effects by inhibiting excessive AMPK activation and subsequent MCU phosphorylation at serine 57. The present study suggests that harpagide regulates mitochondrial calcium homeostasis via the AMPK-MCU phosphorylation cascade, providing a reliable strategy for therapies that target ischemic stroke. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience harpagide ischemic stroke Mitochondrial calcium homeostasis MCU AMPK Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementalData.docx originalwesternblots.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Feb, 2026 Reviews received at journal 20 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviews received at journal 19 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviews received at journal 18 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers invited by journal 18 Feb, 2026 Editor assigned by journal 18 Feb, 2026 Editor invited by journal 06 Feb, 2026 Submission checks completed at journal 03 Feb, 2026 First submitted to journal 03 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Harpagide has been shown to inhibit mitochondrial calcium uptake, but the mechanism remains unclear. In this study, a male ICR mouse model of transient middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia and PC12 and SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) were used. Apoptosis, lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), and the ATP levels were measured to assess the neuroprotective effects of harpagide. DCFH-DA, JC-1, and Rhod-2A probes were used to analyze the mitochondrial function. RT-qPCR and western blot were used to determine the mitochondrial calcium uniporter (MCU), the apoptosis-inducing factor (AIF), Endo G, Cyt C, Caspase-3, and AMPK-MCU phosphorylation expression levels. Additionally, Longa scoring and triphenyl tetrazolium chloride (TTC) staining were used. Transmission electron microscopy (TEM) was used to examine the mitochondrial ultrastructural changes. 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